The replication of HIV-1 is critically dependent upon the regulatory protein, Rev. Previous studies performed in this laboratory have demonstrated that M10, a transdominant mutant form of Rev which interferes with normal Rev function, prevents viral replication in human lymphocytes without affecting T cell function. We have exploited the therapeutic potential of Rev M10 for the treatment of acquired immunodeficiency syndrome (AIDS), and have developed vectors that express this gene product. Preliminary experiments using human lymphocytes indicate that expression of Rev M10 provides significant protection against HIV infection. A human clinical protocol was subsequently developed which is designed to determine whether Rev M10 will prolong the survival of T cells in HIV-infected individuals. This protocol was approved by the Recombinant DNA Advisory Committee of the NIH and will begin in the near future in adult volunteers. Despite the similarities between adult and pediatric AIDS, there are a number of differences between them which distinguishes their natural history and the course of these diseases. For this reason, we now propose to develop human clinical studies and preclinical models to implement gene therapy in pediatric populations. The first goal is to implement gene therapy in pediatric groups by delivering Rev M10 vectors into the T cells of these patients. We will utilize vectors developed in separate studies, which incorporate different cis-activating regulatory elements to increase the expression of this protein in CD4+ cells. The vectors will be tested on T cells stimulated with anti-CD3/IL2 or anti-CD3/CD28. The second aim for this project will be to develop and evaluate gene transfer in hematopoietic stem cells. These studies will involve the use of bone marrow and/or umbilical cord blood to be transplanted into different subsets of children who have an intact thymus that still retains the ability to reconstitute the immune system. Particle-mediated gene transfer will be developed along with the retroviral vector technology to deliver genes into hematopoietic stem cells. The third aim is to optimize gene expression and analyze the ability of the transduced cells to engraft and persist during development in vivo. Initial studies will be performed in animal models. These essential studies will facilitate the initiation of human gene therapy trials for AIDS in children and allow them to proceed with greater safety and efficacy. At the same time that these studies serve as a paradigm for other gene therapy approaches to the treatment of AIDS, they will also provide fundamental information on the pathogenesis of AIDS in children.